Alzheimer's disease is a common, chronic neurodegenerative disease, characterized by a progressive loss of memory and sometimes severe behavioral abnormalities, as well as an impairment of other cognitive functions that often leads to dementia and death. It ranks as the fourth leading cause of death in industrialized societies after heart disease, cancer, and stroke. The incidence of Alzheimer's disease is high, with an estimated 2.5 to 4 million patients affected in the United States and perhaps 17 to 25 million worldwide. Moreover, the number of sufferers is expected to grow as the population ages.
A characteristic feature of Alzheimer's disease is the presence of large numbers of insoluble deposits, known as amyloid plaques, in the brains of those affected. Autopsies have shown that amyloid plaques are found in the brains of virtually all Alzheimer's patients and that the degree of amyloid plaque deposition correlates with the degree of dementia (Cummings & Cotman, 1995, Lancet 326:1524–1587). While some opinion holds that amyloid plaques are a late stage by-product of the disease process, the consensus view is that amyloid plaques are more likely to be intimately, and perhaps causally, involved in Alzheimer's disease.
A variety of experimental evidence supports this view. For example, Aβ, a primary component of amyloid plaques, is toxic to neurons in culture and transgenic mice that overproduce Aβ in their brains show significant deposition of Aβ into amyloid plaques and significant neuronal toxicity (Yankner, 1990, Science 250:279–282; Mattson et al., 1992, J. Neurosci. 12:379–389; Games et al., 1995, Nature 373:523–527; LaFerla et al., 1995, Nature Genetics 9:21–29). Mutations in the APP gene, leading to increased Aβ production, have been linked to heritable forms of Alzheimer's disease (Goate et al., 1991, Nature 349:704–706; Chartier-Harlan et al., 1991, Nature 353:844–846; Murrel et al., 1991, Science 254:97–99; Mullan et al., 1992, Nature Genetics 1:345–347). Presenilin-1 (PS1) and presenilin-2 (PS2) related familial early-onset Alzheimer's disease (FAD) shows disproportionately increased production of Aβ1-42, the 42 amino acid isoform of Aβ, as opposed to Aβ1-40, the 40 amino acid isoform (Scheuner et al, 1996, Nature Medicine 2:864–870). The longer isoform of Aβ is more prone to aggregation than the shorter isoform (Jarrett et al, 1993, Biochemistry 32:4693–4697). Injection of the insoluble, fibrillar form of Aβ into monkey brains results in the development of pathology (neuronal destruction, tau phosphorylation, microglial proliferation) that closely mimics Alzheimer's disease in humans (Geula et al., 1998, Nature Medicine 4:827–831). See Selkoe, 1994, J. Neuropathol. Exp. Neurol. 53:438–447 for a review of the evidence that amyloid plaques have a central role in Alzheimer's disease.
Aβ, a 39–43 amino acid peptide derived by proteolytic cleavage of the amyloid precursor protein (APP), is the major component of amyloid plaques (Glenner & Wong, 1984, Biochem. Biophys. Res. Comm. 120:885–890). APP is actually a family of polypeptides produced by alternative splicing from a single gene. Major forms of APP are known as APP695, APP751, and APP770, with the subscripts referring to the number of amino acids in each splice variant (Ponte et al., 1988, Nature 331:525–527; Tanzi et al., 1988, Nature 331:528–530; Kitaguchi et al., 1988, Nature 331:530–532). APP is membrane bound and undergoes proteolytic cleavage by at least two pathways. In one pathway, cleavage by an enzyme known as α-secretase occurs while APP is still in the trans-Golgi secretory compartment (Kuentzel et al., 1993, Biochem J. 295:367–378). This cleavage by α-secretase occurs within the Aβ portion of APP, thus precluding the formation of Aβ. In another proteolytic pathway, cleavage of the Met671-Asp672 bond (numbered according to the 751 amino acid protein) by an enzyme known as β-secretase occurs. This cleavage by β-secretase generates the N-terminus of Aβ. The C-terminus is formed by cleavage by a second enzyme known as γ-secretase. The C-terminus is actually a heterogeneous collection of cleavage sites rather than a single site since γ-secretase activity occurs over a short stretch of APP amino acids rather than at a single peptide bond. Peptides of 40 or 42 amino acids in length (Aβ1-40 and Aβ1-42, respectively) predominate among the C-termini generated by γ-secretase. Aβ1-42 is more prone to aggregation than Aβ1-40, is the major component of amyloid plaque (Jarrett et al., 1993, Biochemistry 32:4693–4697; Kuo et al., 1996, J. Biol. Chem. 271:4077–4081), and its production is closely associated with the development of Alzheimer's disease (Sinha & Lieberburg, 1999, Proc. Natl. Acad. Sci. USA 96:11049–11053). The bond cleaved by γ-secretase appears to be situated within a transmembrane domain of APP. It is unclear as to whether the C-termini of Aβ1-40 and Aβ1-42 are generated by a single γ-secretase protease with sloppy specificity or by two distinct proteases. For a review that discusses APP and its processing, see Selkoe, 1998, Trends Cell. Biol. 8:447–453.
Much interest has focused on the possibility of inhibiting the development of amyloid plaques as a means of preventing or ameliorating the symptoms of Alzheimer's disease. To that end, a promising strategy is to inhibit the activity of β- and γ-secretase, the two enzymes that together are responsible for producing Aβ. This strategy is attractive because, if the formation of amyloid plaques as a result of the deposition of Aβ is a cause of Alzheimer's disease, inhibiting the activity of one or both of the two secretases would intervene in the disease process at an early stage, before late-stage events such as inflammation or apoptosis occur. Such early stage intervention is expected to be particularly beneficial (see, e.g., Citron, 2000, Molecular Medicine Today 6:392–397).
To that end, various assays have been developed that are directed to the identification of compounds that may interfere with the production of Aβ or its deposition into amyloid plaques. U.S. Pat. No. 5,441,870 is directed to methods of monitoring the processing of APP by detecting the production of amino terminal fragments of APP. U.S. Pat. No. 5,605,811 is directed to methods of identifying inhibitors of the production of amino terminal fragments of APP. U.S. Pat. No. 5,593,846 is directed to methods of detecting soluble Aβ by the use of binding substances such as antibodies. Esler et al., 1997, Nature Biotechnology 15:258–263 described an assay that monitored the deposition of Aβ from solution onto a synthetic analogue of an amyloid plaque. The assay was suitable for identifying compounds that could inhibit the deposition of Aβ. However, this assay is not suitable for identifying substances, such as inhibitors of γ-secretase, that would prevent the formation of Aβ. Thus, the assay of Esler is directed to a step that is further along in the formation of amyloid plaque than is the assay described in this application.
Various groups have cloned and sequenced cDNA encoding a protein that is believed to be β-secretase (Vassar et al., 1999, Science 286:735–741; Hussain et al., 1999, Mol. Cell. Neurosci. 14:419–427; Yan et al., 1999, Nature 402:533–537; Sinha et al., 1999, Nature 402:537–540; Lin et al., 2000, Proc. Natl. Acad. Sci. USA 97:1456–1460) but the identity of γ-secretase has been more elusive. A pair of proteins known as presenilin-1 and presenilin-2 are viewed as possible candidates (Selkoe & Wolfe, 2000, Proc. Natl. Acad. Sci. USA 97:5690–5692).
Presenilin-1 (PS1) and presenilin-2 (PS2) are polytopic membrane proteins that are involved in γ-secretase-mediated processing of APP. The most common cause of familial early-onset Alzheimer's disease is the autosomal dominant inheritance of assorted mutations in the PSi gene (Sherrington et al., 1995, Nature 375:754–760). These PSi mutations lead to increased production of Aβ1-42 (Scheuner et al., 1996, Nature Medicine 2:864–870; Duff et al., 1996, Nature 383:710–713; Borchelt et al., 1996, Neuron 17:1005–1013). Similarly, certain mutations in PS2 cause familial early-onset Alzheimer's disease and increased generation of Aβ1-42 (Levy-Lahad et al., 1995, Science 269:970–973). Cultured isolated neurons from PS1-deficient mice exhibit reduced γ-secretase-mediated cleavage of APP (De Strooper et al., 1998, Nature 391:387–390). It was suggested that PS1 might influence trafficking of APP and/or γ-secretase or it might play a more direct role in proteolytic cleavage of APP. Directed mutagenesis of two conserved transmembrane-situated aspartates in PS1 was shown to inactivate γ-secretase activity in cellular assays, suggesting that PS1 is either a required diaspartyl cofactor for γ-secretase or is itself γ-secretase, an intramembranous aspartyl protease (Wolfe et al., 1999, Nature 398:513–517). Moreover, Li et al., 2000, Nature 405:689–694 made photoactivatable derivatives of a highly specific and potent aspartyl protease transition state analog inhibitor and found that the inhibitor selectively labeled presenilin fragments.
Despite results such as those described above, it is still uncertain whether PS1 and PS2 are responsible for the γ-secretase activity that is relevant to the processing of APP in connection with Alzheimer's disease. It is desirable to identify all the proteases that may have γ-secretase activity and thus may be involved in the development of Alzheimer's disease. Therefore, the identification and purification of novel proteins possessing γ-secretase activity is valuable. The availability of such novel proteases would allow for the development of assays to discover inhibitors of such proteases. Such inhibitors are likely to be valuable in the treatment of Alzheimer's disease.